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THERMAL STRESS AND THE HEAT

SHOCK RESPONSE

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Contents Types of Microbes on the basis of Temperature

Thermophile

Psychrophile

Mesophile

Thermal Stress

Heat Shock Response

Description

Types of Heat Shock Responses

Heat Shock Response against Cytoplasmic Thermal

Stress

σᴴ Regulation

Heat Shock Response against Periplasmic Thermal

Stress

σᴱ Regulation

Cpx systems

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THERMAL STRESS AND THE HEAT SHOCK RESPONSE

As with pH, microbes exhibit a wide range of temperatures at which they can grow.

Types of Microbes on the basis of Temperature

There are three types of microbes on the basis of temperature.

1. Thermophiles

2. Psychrophiles

3. Mesophiles

Thermophile

A thermophile is an organism — a type of extremophile — that thrives at

relatively high temperatures, between 45 and 122 °C (113 and 252 °F).

Many thermophiles are archaea. Thermophilic eubacteria are suggested to have

been among the earliest bacteria.

Thermophiles are found in various geothermally heated regions of the Earth, such

as hot springs like those in Yellowstone National Park and deep sea hydrothermal

vents, as well as decaying plant matter, such as peat bogs and compost.

As a prerequisite for their survival, thermophiles contain enzymes that can

function at high temperatures. Some of these enzymes are used in molecular

biology (for example, heat-stable DNA polymerases for PCR), and in washing

agents.

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"Thermophile" is derived from the Greek: θερμότητα (thermotita),

meaning heat, and Greek: υίλια (philia), love

A colony of thermophiles in the outflow of Mickey Hot Springs, Oregon, the water temperature is approximately 60°C.

Psychrophile

Psychrophiles or cryophiles are extremophilic organisms that are capable of

growth and reproduction in cold temperatures, ranging from −15°C to +10°C.

Temperatures as low as −15°C are found in pockets of very salty water (brine)

surrounded by sea ice.

They can be contrasted with thermophiles, which thrive at unusually hot

temperatures. The environments they inhabit are ubiquitous on Earth, as a large

fraction of our planetary surface experiences temperatures lower than 15°C.

They are present in alpine and arctic soils, high-latitude and deep ocean waters,

polar ice, glaciers, and snowfields.

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Examples are Arthrobacter sp., Psychrobacter sp. and members

of the genera Halomonas, Pseudomonas, Hyphomonas and

Sphingomonas.

Cold-loving extremophile bacteria

Mesophile

A mesophile is an organism that grows best in moderate temperature, neither too

hot nor too cold, typically between 20 and 45 °C (68 and 113 °F). The term is

mainly applied to microorganisms.

The habitats of these organisms include especially cheese, yogurt, and mesophile

organisms are often included in the process of beer and wine making.

Organisms that prefer cold environments are termed psychrophilic, those

preferring warmer temperatures are termed thermophilic and those thriving in

extremely hot environments are hyperthermophilic. All bacteria have their own

optimum environmental surroundings and temperatures in which they thrive the

most.

Examples include E.coli and Streptococcus.

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Lactic acid bacteria

Escherichia coli

Thermal Stress

The stress induced in the body of microbes due to high temperature is called thermal

stress.

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Heat Shock Response

The heat shock represents a ubiquitous protective and homeostatic cellular response to

cope with heat-induced damage in proteins.

Description

Upon a shift from 30◦ to 42 ◦C, E. coli and other bacteria transiently increase the

rate of synthesis of a set of proteins called heat shock proteins (HSPs).

Many of these HSPs are required for cell growth or survival at more elevated

temperatures (thermotolerance).

Among the induced proteins are DnaJ and DnaK, the RNA polymerase σ70

subunit (rpoD), GroES, GroEL, Lon protease, and LysU.

There are nearly 50 heat shock–inducible proteins identified in E. coli.

Types of Heat Shock Responses

There are two types of heat shock responses.

1. Heat shock response against cytoplasmic thermal stress

2. Heat shock response against periplasmic thermal stress

The hallmark ofthe Gram-negative cell is the existence of two membrane-bound

subcellular compartments:

Cytoplasm

Periplasm

Conditions in each of these compartments differ markedly.

The cytoplasm is energy rich, reducing (low redox potential), and osmotically

stable.

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Whereas the periplasm lacks ATP, is oxidizing, and is in contact withthe

external milieu.

Since optimal cell growth requires that the cell senses and responds to changes in these

disparate sub cellular compartments, it is not surprising that the stress responses in E. coli

and S. typhimurium are compartmentalized into cytoplasmic and extracytoplasmic

responses.

1. Heat Shock Response against

Cytoplasmic Thermal Stress

The σᴴ regulon provides protection against cytoplasmic thermal stress.

The E. coli rpoH locus (formerly called htpR) encodes a 32 kDa σ factor,

alternatively called σᴴ or σ³², which redirects promoter specificity of RNA

polymerase.

The σᴴ protein regulates the expression of 34 heat shock genes.

The simple explanation for how heat shock increases expression of the σᴴ regulon

is that heat shock first causes an elevation in σᴴ levels, which in turn increases

expression of the σᴴ target genes.

Although the principle is simple, the controls governing σᴴ production are

complex.

First, a temperature upshifts from 30 ◦ to 42 ◦C results in the increased translation

of rpoH message.

Cis-acting mRNA sites within the 5’ region of rpoH message form temperature-

sensitive secondary structures that sequester the ribosome-binding site. At higher

temperatures, these secondary structures melt, thereby enabling more efficient

translation of the rpoH message.

In addition to the increased translation of rpoH message, the σᴴ protein itself

becomes more stable, at least transiently.

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The mechanism regulating proteolysis centers on whether σᴴ associates with RNA

polymerase. During growth at 30 ◦C, σᴴ can be degraded by several proteases

including FtsH, HslVU, and ClpAP.

However, if σᴴ is bound to RNAP, σᴴ is protected from degradation.

The cell uses the DnaK-DnaJ-GrpE chaperone team to interact with σᴴ at low

temperature, sequestering σᴴ from RNA polymerase.

Failure to bind RNAP facilitates degradation of the σ factor.

Upon heat shock, there is an increase in the number of other unfolded or denatured

proteins that can bind to DnaK or DnaJ. This reduces the level of free DnaK/DnaJ

molecules available to bind σᴴ, allowing σᴴ tobind RNAP, which protects σᴴ from

degradation.

As the cell reaches the adaptation phase following heat shock, the levels of DnaK

and DnaJ rise (both are induced by σᴴ) and can again bind σᴴ, redirecting it toward

degradation. Nevertheless, even though σᴴ degradation resumes, translation of

rpoH remains high at the elevated temperature and σᴴ continues to accumulate,

although at a slower rate.

In addition to translational and proteolytic controls, production of σᴴ is regulated at the

transcriptional level via a feedback mechanism. There are four promoters driving rpoH

expression, three of which are dependent on σ70, the housekeeping σ factor.

The gene encoding σ70, rpoD, is also a heat shock gene induced by σᴴ. So increased

production of σᴴ increases σ70, which increases transcription of rpoH. The fourth rpoH

promoter is recognized by another σ factor, σᴱ, encoded by rpoE.

The heat shock response is also triggered by a variety of environmental agents such as

ethanol, UV irradiation, and agents that inhibit DNA gyrase. Induction by all of these

stimuli occurs through σᴴ. The only explanation that appears reasonable is the

accumulation of denatured or incomplete peptides. There is a potential alarmone that has

been implicated in signaling expression of this global network. The molecule is

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diadenosine 5_, 5___-P1, P4-tetraphosphate (AppppA), which is made by some

aminoacyl–tRNA synthetases (e.g., lysU) at low tRNA concentrations. How this may

influence the response is not known.

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2. Heat Shock Response against

Periplasmic Thermal Stress

The extra cytoplasmic response pathways involve two partially overlapping signal

transduction cascades:

I. σᴱ regulation

II. Cpx systems

These pathways are induced following the accumulation of misfolded proteins in the

periplasmas a result of stresses such as high temperature, pH extremes, or carbon/energy

starvation.

I. σᴱ Regulation

σᴱis a member of the extracytoplasmic function (ECF) subfamily of σ factors.In E. coli,

σᴱ is responsible for the transcription of up to genes, including:

rpoH (σᴴ)

degP (htrA) encoding a periplasmic protease for the degradation of

misfolded proteins

fkpA encoding a periplasmic peptidyl prolyl isomerase

rpoE rseABC operon

The gene encoding σᴱ, rpoE, is the first member of an operon followed by the genes rseA,

B, and C (rse, meaning regulators of σE).

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RseA is a transmembrane protein whose cytoplasmic C-terminal domain interacts

with σᴱ, acting as an anti-σfactor.

The periplasmic face of RseA binds to the periplasmic RseB.

Extracellular stress in some way signals increased proteolysis of RseA by the

periplasmic protease DegS, thus relieving the anti-σ effect of RseA on σE.

It has been proposed that Rse Band perhaps other periplasmic proteins involved in

protein folding protect RseA from degradation by binding to the RseA periplasmic

domain, capping the target site of DegS.

Stress-induced misfolding of periplasmic proteins would titrate the RseA cap

proteins off of RseA, rendering the anti-σ factor vulnerable to attack by DegS.

The result would be increased activity of σᴱ leading to increased levels of σᴱ

protein and RseA anti-σ (since they form an operon).

The increased amount of σᴱ will drive further expression of genes whose products

handle the periplasmic damage while the of RseA will enable the cell to down

regulate the system once the capping proteins are again free to bind and protect

RseA from DegS degradation.

II. Cpx systems

A second system dedicated to protecting the periplasmic perimeter of the cellis the

CpxRA two-component system with:

CpxA playing the role of membrane-localized sensor histidine kinase

CpxR as the cytoplasmicresponse regulator

CpxA responds to envelope stress by autophosphorylation followed by

phosphotransfer to CpxR. CpxR∼P activates expression of:

dsbA (disulfideoxidoreductase)

ppiA and ppiD encoding peptidyl-prolyl-isomerases

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In E. coli ,in conjunction with σE, degP (htrA)

In addition, CpxR∼P activates transcription of cpxP encoding a small protein that

negatively regulates the CpxAR regulon, probablyby binding to a periplasmic

domain of CpxA.

The ability to autoactivate and then repress (via CpxP) enables a temporary

amplification of the Cpx response that maybe important to rescue cells from transitory

stresses.

PrpA and PrpB are type Iserine/threonine phosphatases that also participate in the ECF

pathway at least inpart by affecting the phosphorylation level of CpxR.

B. subtilis has four classes of heat shock genes. One is particularly interesting because

of the mechanism used to control the genes. Class I heat shock genes include the major

chaperones DnaK-DnaJ-GrpE and GroEL-GroES. Their transcription requires the

housekeeping σ factor, σA (σ70) and is negatively controlled by a repressor called HrcA.

HrcA binds to a class I geneoperator, a well-conserved 9 bp inverted repeat with a 9 bp

spacer, called CIRCE.

TheCIRCE/HrcA regulon is normally repressed by the HrcA repressor but can be heat

induced by inactivating the repressor. The molecular switch involves the chaperone

GroE. Unlike σ³² in E. coli, the GroE chaperones bind to and facilitate folding of HrcA

and thereby modulate repressor function. Titration of GroE by stress-induced misfolded

proteins results in lower HrcA repressor activity.